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CXXIV. THE PRODUCTION OF ORGANIC COMPOUNDS OF SULPHUR IN BACTERIAL CULTURES WITH SPECIAL REFERENCE TO GLUTATHIONE.

By JAMES WALTER MCLEOD AND JOHN GORDON.
From the Department of Pathology and Bacteriology, Leeds University.

(Received July 26th, 1924.)
THERE is an extensive literature which deals with the production of sulphides by bacteria. Without attempting a detailed review of this literature it will be sufficient to draw attention to some of the more important facts which have been established. . It seems to be clear that a number of bacteria are able to produce H S from media containing sulphur, sulphite or cystine; but that H28 is rarely produced from taurine or sulphates. Further, the importance of cystine as a source of sulphide has been demonstrated. Sulphide is produced when bacteria are cultivated in synthetic media containing cystine but no other source of sulphur [Sasaki and Otsuka, 1912; Myers, 1920]. As sources of sulphide the different peptones appear to vary considerably and even from sample to sample, when used in culture media for the demonstration of sulphide production by bacteria. Certain results, however, have been constantly obtained with different media and by different observers; such are active sulphide production by B. paratyphosus B., B. enteritidis, B. proteus, B. cloacae, B. typhosus and absence of sulphide production by B. paratyphosus A and the dysentery bacilli [Myers, 1920; Thompson, 1921; Tilley, 1923]. The formation of volatile sulphides by anaerobes has been frequently noted [Med. Res. Council, 1919]. It is interesting that Thompson observes a general although not invariable rule that bacteria in the coliform group which ferment lactose do not form sulphide. Tanner notes that yeasts possess much greater power than bacteria for reducing both organic and inorganic compounds of sulphur. The observations with regard to the formation of organic compounds of sulphur by bacteria are not numerons. Sasaki and Otsuka [1912], working with pure cultures and synthetic media, were unable to demonstrate mercaptan formation. However, Kondo [1923], working with synthetic media contaiig I-cystine, showed that B. proteus constantly and B. coli occasionally produced mercaptan provided one of the following were present in the medium glucose, lactose, saccharose, glycerol or histidine. No other amino acid tested could take the place of histidine. He also obtained presumptive evidence of the production of ethyl sulphide or some other alkyl sulphide. H2S was invariably produced under these conditions.

Reactions were invariably negative with Gonococcus. proteus. gaertner and other strains allied to B. In observations with cultures of bacteria which could grow aerobically tests were made both with and without previous reduction. The strongest positive reactions were obtained with cultures of B. and of the cholera vibrio. weaker positives were obtained with B. Reduction was effected by an aluminium mercury couple. the reactions with the more strict anaerobes appeared to be the stronger. of broth culture. grown under anaerobic conditions were apparently a good deal. such compounds can only account for a small fraction of the reacting substances. and with some strains of B. Some part of this reaction may be due to inorganic sulphides. it seemed to us to be desirable to get more extended information with regard to the occurrence of substances reacting with nitroprusside in bacterial cultures. McLEOD AND J. then 10 cc. 1922] that traces of H202 were formed in the course of oxidations in which glutathione acted as an accelerator of respiration in the presence of oxygen. Staphylococcus albus. These observations were limited to the occurrence of the nitroprusside reaction in cultures of such bacteria as he had tested. paratyphosus A. and Shiga strains of dysentery bacilli and with B. typhosus and with B. of a 5 % solution of nitroprusside and lastly 5-7 cc. All the cultures of anaerobes tested gave strong reactions and in so far as could be judged with a method. Doubtful results were got with some strains of Staphylococcus aureus. 1922]. of strong NH40H were added. We hoped to establish some correlation between production by bacteria of H202 on the one hand and of substances reacting with nitroprusside on the other.938 J. coli. 0-5 cc. with B. negative with others. Meningococcus. The nitroprusside reactions obtained with cultures of anaerobes or with cultures of B. W. Pneumococcus and strains of Influenza bacilli. The method of carrying out the reaction was as follows: a test-tube was filled to the depth of half an inch with (NH4)2S04 crystals. Y. Hopkins and Dixon. At all events no mention is made of the isolation of the compound from bacterial cultures. paratyphosus B. As we were engaged about that time in an investigation of the production of 11202 by bacteria [McLeod and Gordon. which is not particularly well adapted to quantitative determinations. paratyphosus B. paratyphosus B. GORDON In view of the rather scanty existing evidence for the production of organic compounds of sulphur by bacteria it was of particular interest that Hopkins in the account published in 1921 of his important discovery of the compound glutathione referred to its probable occurrence in bacterial cultures. Streptococci (haemolytic and non-haemolytic). It was considered probable by Hopkins [1921. Flexner.stronger than those got on reducing the original broth for 5-10 minutes with an active HgAl couple with a view to reducing the oxidised glutathione from the meat extract. mercaptans or acetone but inasmuch as the reaction is little or not at all decreased by prolonged boiling. .

3-1 hour. the MnO2 being subsequently removed by filtration. The accuracy of this supposition was tested in two ways. Both B. with an active couple. paratyphosus B. however. fresh or reduced. per 1000 of H202. It was found that broth reduced for 1 hour by an active couple gave a reaction little if at all inferior to that obtained in the same broth after the growth of an anaerobe. Wolffenstein [1894] draws attention to the fact that the liability of H202 to destruction by heating has been exaggerated and shows that in the absence of (a) traces of heavy metals. We have therefore been led to conclude that bacteria do not produce glutathione when cultivated in nutrient broth and that the nitroprusside reactions observed in bacterial cultures are mainly or entirely due to the . This substance. It was not often possible however to destroy the residual H202 completely by subsequent boiling of the broth. The broth resulting from each of those three forms of treatment was proved to be free from glutathione as it gave no nitroprusside reaction after prolonged reduction. were successfully cultivated in all three media although the growths were distinctly less copious than in the original broths. that a heavy suspension of a surface growth of B. presumably glutathione. welchii and B.ORGAXIC SULPHUR COMPOUNDS IN B3ACTERIAL CULTURES 939 It was found. (3) Shaking with a heavy emulsion of staphylococci rich in catalase [McLeod and Gordon. Cultures of these bacteria in the same broth subjected to the same treatment with staphylococci but not oxidised by 1202 gave frank nitroprusside reactions. We therefore had recourse to three different methods of getting rid of the residual H202. The reductions to which reference is made in the earlier part of the paper were not carried out for more than 10-15 minutes. and (c) solid particles of any kind. These were: (1) Shaking with MnO2 powder. never gave a definite nitroprusside reaction. Such cultures always failed to give any trace of nitroprusside reaction. R. whether boiled or unboiled. The second way of testing our theory was to observe whether by means of prolonged reductions by aluminium merculy couples it was possible to produce reactions in ordinary meat extract broth similar in intensity to those observed in bacterial cultures. the first was an investigation of the power of bacteria to produce a nitroprusside reaction in broth freed from substances reacting with nitroprusside. can be destroyed by heating with 1-2 parts. (2) Warming with 0*5-1 % of glucose in the presence of a trace of FeSO4. welchii on a solid medium. (b) substances of alkaline reaction. 1923] and filtering off the staphylococci by meahs of a porcelain candle (Maassen type). it is relatively heat stable. This and some experimental work in which old and recent yeast extracts were compared with bacterial cultures in regard to rates of disappearance of the nitroprusside reaction led us to wonder whether the nitroprusside reaction occurring in bacterial cultures was not entirely due to a very effective reduction of the oxidised glutathione originally present in the medium.

499. The last subject we hope to deal with in a subsequent communication. J. Pneumococcus. Amer. proteus. B. or of the dysentery bacilli (Flexner. J. . Chem. B. Hopkins and Dixon (1922). W. and Shiga types).940 J. REFERENCES. Biochem. 42. paratyphosus A. 5. Streptococcus. (4) The thermo-stable substance reacting with nitroprusside detected in bacterial cultures is not produced by the bacteria but is the oxidised glutathione or some related compound originally present in the medium and reduced by the bacteria in the course of their growth. J. Tilley (1923). J. (1) A distinct nitroprusside reaction has been observed in 24-48 hour cultures of the following bacteria-all anaerobes tested (six. Cholera vibrio (one strain). 286. Meningococcus.euch. B. B.. 39. MaLEOD AND J. 383. 198. B. Med. influenzae.(1923). Sasaki and Otsuka (1912). Medical Research Council (1919). Chem. some strains of B. 287. Morax-Axenfeld. Ges. Bact. 8. A point of some importance arising from these observations is the marked permanence of glutathione in the oxidised condition in media prepared from meat extract and the possibility that it is one of the important constituents of meat extract for promoting the growth of certain bacteria. 54. including B. Biochem. Special Report Series. Thompson (1921). 77. 663. Research. 16. Bact. 231. (3) Where a facultative anaerobe gives a nitroprusside reaction it is much stronger if the bacterium is grown in anaerobic or nearly anaerobic conditions. Hopkins (1921). paratyphosus B. J. 527. coli and B. Glutathione would not appear to play any part in the production of H202 by bacteria of the streptococcal group although it may in the case of the anaerobes. J. GORDON special aptitude of the bacteria concerned for reducing the residual oxidised glutathione present in the medium. and most allied species. Biochem. chem. 326. Bact. tetani and Vibrion septique). SUMMARY. (2) No distinct nitroprusside reactions have been observed in cultures of Staphylococcus. Soc. Gonococcus. pyocyaneus. 63. Y. Z. Tanner (1918). Myers (1920). Biol. 40. 15. B. Wolifenstein (1894). (6) Bacteria capable of reducing glutathione probably utilise it when growing under anaerobic conditions to accelerate the oxidations necessary to their metabolism. McLeod and Gordon (1922). The nitroprusside reaction in a bacterial culture in meat extract broth is therefore an indication of the reducing powers of that bacterium and very possibly an indication of its capacity to utilise glutathione in carrying out some of the oxidations required for its metabolism. J. B. We have pleasure in acknowledging our indebtedness to the Medical Research Council for a grant in aid of this work. Ber. 26. Biochem. J. . (5) Bacteria therefore do not produce glutathione or substances allied to it reacting with nitroprusside. d. 136. 39. Kondo (1923). 208. typhosus. Path. 115. welchii. 3307. Z.